Abstract: The goal of this research is to elucidate molecular mechanisms by which Ca2+, intermolecular cooperation, and protein phosphorylations regulate contraction of mammalian heart muscle. Experiments will test specific hypotheses regarding the regulation of tension and the rate of tension development in skinned myocytes from mouse and rat. Mechanisms of these effects will be studied by altering the phosphorylation states and subunit composition of accessory and regulatory proteins in both the thick and thin filaments, and in some cases replacing them with mutant proteins with altered functional proteins. (1) We hypothesize that myosin regulatory light chain (RLC) regulates the availability of myosin to actin by influencing the flexibility of the myosin head/rod junction, i.e., RLC normally limits the availability of myosin to actin by influencing the flexibility of the myosin head/rod junction, i.e., RLC normally limits the availability of cross bridges to actin, but this repression is relieved by RLC extraction, Ca2+ binding to RLC or RLC phosphorylation. This idea will be tested by studying the functional consequences due to (i) RLC knock-out in mice or in cardiac myocytes from cultured ES cells, (ii) mutations of RLC, or (iii) expression of mutant RLC. Underlying mechanisms will be studied by measuring Ca2+ binding to RLC and using x-ray scattering to assess structural changes in RLC. (2) We hypothesize that phosphorylation of myosin binding protein-C contributes to myocyte contractile responses to agonists that activate sub-cellular PKA and PKC pathways by modifying cross-bridge interaction kinetics. Gene knock-out and mutation will be used to investigate the roles of MyBP-C in stretch activation of myocardium and the contractile responses of skinned cardiac myocytes to PKA and PKC. (3) We propose that thin filament regulatory proteins, troponin T (TnT) and tropomyosin (Tm) contribute to the cooperative mechanisms of thin filament activation in myocardium. Transgenic mice expressing alternate, isoforms of TnT and Tm in the heart will be used to investigate the roles of these proteins in the cooperative binding of Ca2+ by TnC and the cooperative activation of the thin filament by strong binding cross- bridges. Results from this study should provide new information about mechanisms by which contractile state is normally altered in myocardium and mechanisms underlying functional deficits in diseased hearts.